U.S. patent application number 11/061018 was filed with the patent office on 2005-08-25 for method of detecting error spot in dna chip and system using the method.
Invention is credited to Oh, Ji-young.
Application Number | 20050186670 11/061018 |
Document ID | / |
Family ID | 34747931 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186670 |
Kind Code |
A1 |
Oh, Ji-young |
August 25, 2005 |
Method of detecting error spot in DNA chip and system using the
method
Abstract
Provided are a method of detecting an error spot and a system
using the method. The method includes analyzing a difference in
variances of a background intensity and a foreground intensity for
each spot in a DNA chip, verifying whether the mean of the
background intensity and the mean of the foreground intensity are
significantly different from each other based on the difference in
variances, and judging an error spot based on the results of the
verifying operation. Thus, the reliability in statistical analysis
can be increased by excluding the error spot in the statistical
analysis.
Inventors: |
Oh, Ji-young; (Suwon-si,
KR) |
Correspondence
Address: |
CANTOR COLBURN, LLP
55 GRIFFIN ROAD SOUTH
BLOOMFIELD
CT
06002
|
Family ID: |
34747931 |
Appl. No.: |
11/061018 |
Filed: |
February 18, 2005 |
Current U.S.
Class: |
435/287.2 ;
356/139.04; 369/53.13 |
Current CPC
Class: |
G16B 25/00 20190201 |
Class at
Publication: |
435/287.2 ;
369/053.13; 356/139.04 |
International
Class: |
G11B 007/00; G01B
011/26; C12M 001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 2004 |
KR |
10-2004-0011654 |
Claims
1. A method of detecting an error spot, comprising the operations
of: analyzing a difference in variances for a background intensity
and a foreground intensity for each spot in a DNA chip; verifying
if a mean of the background intensity and a mean of the foreground
intensity are significantly different from each other, based on the
difference in variances; and judging an error spot based on the
results of the verifying operation.
2. The method of claim 1, wherein the operation of analyzing the
difference in variances comprises performing an f-test based on
each standard deviation of the background intensity and the
foreground intensity.
3. The method of claim 1, wherein the operation of verifying the
means comprises performing a pooled t-test or a non-pooled t-test,
based on the difference in variances.
4. The method of claim 1, wherein the operation of verifying the
means comprises increasing degrees of freedom if the difference in
variances is high.
5. The method of claim 1, wherein the operation of judging an error
spot is based on a p-value calculated from the results of the
operation of verifying the significant difference of the means.
6. The method of claim 5, wherein in the operation of judging the
error spot, a spot is judged as the error spot if the p-value is at
least 0.05.
7. The method of claim 1, further comprising the operation of
receiving resultant files generated from a quantifying process and
parcing the resultant files to extract input data which are
necessary in the operations of analyzing the difference in
variances and verifying the means.
8. The method of claim 7, wherein the input data include a first
mean and a first standard deviation of the background intensity,
the number of pixels in the background, a second mean and a second
standard deviation of the foreground intensity, and the number of
pixels in the foreground.
9. A system for detecting an error spot, comprising: a variance
analysis part for analyzing a difference in variances for
background intensity and a foreground intensity for each spot in a
DNA chip; a mean verifying part for verifying whether a mean of the
background intensity and a mean of the foreground intensity are
significantly different from each other, based on the difference in
variances; and an error spot judging part for judging an error spot
based on the results of the verifying operation.
10. The system of claim 9, further comprising a data input part for
receiving resultant files generated from a quantifying process and
parcing the resultant files to extract input data which are
necessary in the operations of analyzing the difference in
variances and verifying the means.
11. The system of claim 10, wherein the input data include a first
mean and a first standard deviation of the background intensity,
the number of pixels in the background, a second mean and a second
standard deviation of the foreground intensity, and the number of
pixels in the foreground.
12. The system of claim 9, wherein the variance analysis part
analyzes the difference in variances by performing an f-test based
on each standard deviation of the background intensity and the
foreground intensity.
13. The system of claim 9, wherein the mean verifying part verifies
the significant difference of the means by performing a pooled
t-test or a non-pooled t-test based on the difference in
variances.
14. The system of claim 9, wherein the error spot judging part
judges the error spot based on a p-value calculated from the
results in the mean verifying part.
15. A computer-readable recording medium having recorded thereto a
computer program for executing in a computer a method of detecting
an error spot, the method comprising the operations of: analyzing a
difference in variances for a background intensity and a foreground
intensity for each spot in a DNA chip; verifying whether a mean of
the background intensity and a mean of the foreground intensity are
significantly different from each other based on the difference in
variances; and judging an error spot based on the results of the
verifying operation.
16. The method of claim 3, wherein the operation of verifying the
means comprises increasing degrees of freedom if the difference in
variances is high.
Description
BACKGROUND OF THE INVENTION
[0001] This application claims the benefit of Korean Patent
Application No. 10-2004-0011654, filed on Feb. 21, 2004, in the
Korean Intellectual Property Office, the disclosure of which is
incorporated herein in its entirety by reference.
[0002] 1. Field of the Invention
[0003] The present invention relates to a method of detecting an
error spot and a system using the method, and more specifically, to
a method of detecting an error spot by quantifying DNA chips and a
system using the method.
[0004] 2. Description of the Related Art
[0005] DNA chips have been manufactured using molecular biological
technologies and newly developed mechanical and electronic
engineering technologies. DNA chips are chips in which several
hundreds to several hundreds of thousands DNAs are integrated in a
very small space using mechanical automation and electronic control
technologies. That is, DNA chips are chips to which many types of
DNAs are attached with high density for detecting genes. DNA chips
can replace the conventional genetic engineering technologies, such
as southern blotting and northern blotting, mutant detection, and
DNA sequencing.
[0006] DNA chips are classified into four groups depending on the
manufacturing method; pin microarray chips manufactured by micro
dotting (surface contact) using a pin, inkjet chips manufactured by
micro dotting using an inkjet technology, photolithography chips,
and electronic array chips.
[0007] FIG. 1 is a flowchart illustrating a conventional method of
analyzing genes using a DNA chip.
[0008] Referring to FIG. 1, a sample preparation is performed for
taking a sample, i.e., gene to be analyzed (operation (S100)). In
the sample preparation, pure genes to be analyzed are extracted
from a biological sample, such as blood.
[0009] Next, genes extracted via the sample preparation are
amplified to an analyzable level (operation (S110)). The
amplification operation is generally performed by a polymerase
chain reaction (PCR).
[0010] Then, the amplified genes, which are a target sample, are
hybridized in the DNA chip (operation (S120)). In the hybridization
operation, the target sample to be tested is reacted with oligo
samples having information of genes and immobilized on the chip.
Thus the target sample is hybridized with an oligo sample having a
complementary sequence.
[0011] Next, a non-hybridized target sample which remains on the
chip is washed off (operation (S130)). Then, the image of the chip
is scanned by a scanner to detect a degree of hybridization of the
target sample with the oligo probe (operation (S140)).
Subsequently, the scanned image is quantified for a statistical
analysis (operation (S150)).
[0012] After quantifying the image of the DNA chip, a statistical
analysis is performed using various algorithms and the quantified
value of each spot on the chip is analyzed in order to discriminate
whether the target sample is originated from a sick person or a
normal person (operation (S160)).
[0013] As illustrated in FIG. 1, the conventional method of
analyzing genes comprises a series of continuous seven operations.
During experiments between the first operation and the fifth
operation (operations (S100 through S140)), various error factors
and thus various types of error spots are generated. If the
quantification operation is performed based on false information
due to the errors and the statistical analysis is performed using
the quantified false data, the false spot data may reduce a
reliability of the analysis and limit the possibility to identify a
sick person.
SUMMARY OF THE INVENTION
[0014] The present invention provides a method of detecting an
error spot, which increases a reliability in a statistical analysis
by detecting the error spot in a DNA chip and excluding the
detected error spot in the statistical analysis and a system using
the method.
[0015] The present invention also provides a computer-readable
recording medium having recorded therein a computer program for
executing in a computer a method of detecting an error spot, the
method increasing a reliability in a statistical analysis by
detecting the error spot in a DNA chip and excluding the detected
error spot in the statistical analysis.
[0016] According to an aspect of the present invention, there is
provided a method of detecting an error spot, comprising the
operations of: analyzing a difference in variances for a background
intensity and a foreground intensity for each spot in a DNA chip;
verifying if a mean of the background intensity and a mean of the
foreground intensity are significantly different from each other,
based on the difference in variances; and judging an error spot
based on the results of the verifying operation.
[0017] According to another aspect of the present invention, there
is provided a system for detecting an error spot, comprising: a
variance analysis part for analyzing a difference in variances for
background intensity and a foreground intensity for each spot in a
DNA chip; a mean verifying part for verifying whether a mean of the
background intensity and a mean of the foreground intensity are
significantly different from each other, based on the difference in
variances; and an error spot judging part for judging an error spot
based on the results of the verifying operation.
[0018] According to still another aspect of the present invention,
there is provided a computer-readable recording medium having
recorded thereto a computer program for executing in a computer a
method of detecting an error spot, the method comprising the
operations of: analyzing a difference in variances for a background
intensity and a foreground intensity for each spot in a DNA chip;
verifying whether a mean of the background intensity and a mean of
the foreground intensity are significantly different from each
other based on the difference in variances; and judging an error
spot based on the results of the verifying operation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The above and other features and advantages of the present
invention will become more apparent by describing in detail
exemplary embodiments thereof with reference to the attached
drawings in which:
[0020] FIG. 1 is a flowchart illustrating a conventional method of
analyzing genes using a DNA chip;
[0021] FIG. 2 is a flowchart illustrating an image processing
procedure for a DNA chip;
[0022] FIG. 3 is a diagram illustrating an image scanning of a DNA
chip;
[0023] FIG. 4 is a diagram illustrating errors generated during
analyzing a DNA chip and the types of scanning errors corresponding
to the errors generated during analyzing the DNA chip;
[0024] FIG. 5 is a diagram illustrating results generated from the
types of scanning errors in FIG. 4;
[0025] FIG. 6A is a graph illustrating the relationship between a
spot size and a spot intensity;
[0026] FIG. 6B is a graph illustrating the relationship between a
spot intensity and its standard deviation;
[0027] FIGS. 7A and 7B are diagrams illustrating input data used in
a method of detecting an error spot according to an embodiment of
the present invention;
[0028] FIG. 8 is a flowchart illustrating a method of detecting an
error spot according to an embodiment of the present invention;
[0029] FIG. 9 is a block diagram illustrating a system for
detecting an error spot according to another embodiment of the
present invention;
[0030] FIGS. 10 and 11 are diagrams illustrating the ratio and the
type of error spots detected in each DNA chip; and
[0031] FIG. 12 is a diagram illustrating a change of Robust M
caused by excluding error spots.
DETAILED DESCRIPTION OF THE INVENTION
[0032] FIG. 2 is a flowchart illustrating an image processing
procedure for a DNA chip and FIG. 3 is a diagram illustrating an
image scanning of a DNA chip.
[0033] In general, the image processing procedure of a DNA chip
includes a scanning operation and a quantification operation. The
scanning operation and the quantification operation are closely
related to each other. Values obtained from the quantification
operation change depending on a scanning method.
[0034] Referring to FIGS. 2 and 3, there is first performed
addressing of a location and a shape of each spot in the DNA chip
and gridding of a region to be read (operation (S200)).
[0035] Next, a segmentation is performed (operation (210)) in which
pixels belonging to a background region (310) and pixels belonging
to a foreground region (320) in the addressed spot are segmented.
Various methods have been proposed to segment the foreground (320)
and the background (310). Representative methods include a fixed
circle assumption and an adaptive circle assumption.
[0036] The fixed circle assumption method segments a background and
a foreground by plotting identical circles for each spot, on the
assumption that all spots have the same size and shape. The
adaptive circle assumption plots a shape of a spot by connecting
pixels having an intensity remarkably different from adjacent
pixels, by taking it into account that each spot may have a
different shape and a different size.
[0037] After segmenting the background and the foreground
(operation (S210)), a median value of the intensity is read for
each pixel in the background and the foreground, respectively, and
the median values are summed up and then divided by the number of
pixels to obtain a mean of the intensity for the background and the
foreground, respectively. In addition, a standard deviation for the
background and the foreground, respectively, is obtained based on
the median values of the intensity for each pixel.
[0038] Also, various methods of quantifying an intensity by
scanning the spot are disclosed. Representative quantifying methods
include a method using a standard deviation of a background, a
method using a spotted area, and a method using a center point.
[0039] The method using a standard deviation of a background is
performed based on the percentage of pixels in a foreground, a
median intensity of each pixel, which is larger than a median
intensity for a background, being added to one or two times its
standard deviation. This method is sensitive to the standard
deviation of the intensity. However, it is difficult to determine a
critical value of the percentage and discriminate an error in
alignment and a spot shape.
[0040] The method using a spotted area discriminates an error spot
by comparing the area of a foreground with the area of gridded
region in the spot.
Spot shape QC score=(spot area=pR.sup.2/2pR)/(spot
perimeter=R/2)
[0041] If QC score.ltoreq.R/2, the spot is regarded as an error
spot.
[0042] That is, as a result of the above comparison of the areas,
if the area of a foreground is less than R/2, the spot is regarded
as an error spot. However, this method cannot distinguish the
errors, such as intensity error, spot spreading, non-uniformity of
a background and the like.
[0043] The method using the center point of a spot comprises
comparing the differences between the center point of a spot which
was gridded in an immobilized state and the center point of a spot
which was gridded in a flexible state and classifying spots having
a considerable difference as error spots. However, this method
cannot distinguish the errors, such as intensity error, spot
spreading and the like.
[0044] FIG. 4 is a diagram illustrating errors generated during
analyzing a DNA chip and the types of scanning errors corresponding
to the errors generated during analyzing the DNA chip.
[0045] Referring to FIG. 4, the errors (400) generated during
analyzing a DNA chip include (1) low DNA amount in the spot, (2)
purity of DNA, (3) attachment of glass, (4) uneven hybridization,
(5) suboptimal labeling, (6) target 2ndary structures, (7) array
surfaces, (8) dirty pins, (9) spotting liquid volume, (10)
scratched surfaces, (11) uneven coating, (12) bleeding and the
like.
[0046] The types of the corresponding scanning errors generated
from the errors (400) include (1) spot intensity, (2) spot size,
(3) spot morphology, (4) alignment error, (5) bleeding, (6)
background intensity, (7) background noisy and the like.
[0047] FIG. 5 is a diagram illustrating results resulting from the
types of scanning errors as illustrated in FIG. 4.
[0048] Referring to FIG. 5, intensity variation results from the
errors of spot size, spot morphology, alignment error, bleeding,
and background noisy. Low intensity results from the errors of spot
size, spot morphology, alignment error, and bleeding. Further,
saturated intensity results from the errors of spot size, spot
morphology, and bleeding.
[0049] Thus, as a result of analyzing the relationship between the
error types in the DNA chip and results thereof, the error types
are classified as spots exhibiting (1) low intensity, (2) intensity
variation in the foreground and the background, or (3) saturated
intensity.
[0050] FIG. 6A is a graph illustrating the relationship between a
spot size and a spot intensity, and FIG. 6B is a graph illustrating
the relationship between a spot intensity and its standard
deviation.
[0051] Referring to FIG. 6B, the statistical result is that as the
deviation of the intensity is higher, a possibility that the
intensity is low is higher.
[0052] FIGS. 7A and 7B are diagrams illustrating examples of input
data used in a method of detecting an error spot according to an
embodiment of the present invention.
[0053] Referring to FIGS. 7A and 7B, the spots (700) are segmented
into the foreground (720) and the background (710). Then, a
foreground mean (770) is obtained by dividing a median of intensity
of each pixel comprising the foreground (720) by the foreground
pixel number (780). And a foreground standard deviation (775) is
obtained from the foreground mean (770). Also, a background mean
(775) is obtained by dividing a median of intensity of each pixel
comprising the background (710) by the background pixel number
(765). And a background standard deviation (760) is obtained from
the background mean (775).
[0054] Thus, input data (750) used in a method of detecting an
error spot according to an embodiment of the present invention
consist of the mean (770) and the standard deviation (775) for the
foreground intensity and the foreground pixel number (780), and the
mean (755) and the standard deviation (760) for the background
intensity and the background pixel number (765).
[0055] There are many programs for quantifying the spot intensity
of the DNA chip, each program showing a mean, standard deviation,
and pixel number for the background and the foreground,
respectively, as a result of quantification. Thus, if the
quantification is possible using a conventional program, variables
necessary to perform an embodiment of the present invention may be
extracted from the files output as a result of the quantification.
In general, the quantification program outputs files with a GPR
file extension.
[0056] FIG. 8 is a flowchart illustrating a method of detecting an
error spot according to an embodiment of the present invention.
[0057] Referring to FIG. 8, the quantification program produces an
output file including the respective mean, standard deviation, and
pixel number for foreground intensity and background intensity of
the spot. A conventional quantification program can be used in the
embodiment of the present invention.
[0058] The output file is subject to parcing (operation (S800)), in
order to extract input data consisting of the respective mean,
standard deviation, and pixel number for foreground intensity and
background intensity of the spot, which are necessary to the
present invention from the output file.
[0059] Then, the difference in variances is analyzed using the
standard deviations for each foreground intensity and background
intensity, respectively ((operation (S805)). The f-test is used for
analyzing the difference in variances. The f-test is used to verify
whether variances of two groups are significantly different from
each other.
[0060] After the completion of the analysis ((operation (S805)), a
verifying operation is performed to establish whether the mean of
the background intensity and the mean of the foreground intensity
are significantly different from each other, based on the
difference in variances ((operations (S810 through S815)). If the
results of the difference in variances obtained from the f-test are
significant, a pooled t-test is performed for verifying the means.
Contrary to this, if the results of the difference in variances
obtained from the f-test are not significant, a non-pooled t-test
is performed for verifying the means. For example, the resulting
value of at least 0.05 in the f-test is judged as being
significant, and the resulting value of no more than 0.05 in the
f-test is judged as not being significant. The value of 0.05, which
is used as a criterion for the establishing the significance, can
be somewhat changed depending on the results of statistical
results.
[0061] The t-test is used to verify whether means of two groups are
significantly different or not. 1 Equation 1 test statistic t = ( Y
_ 1 - Y _ 2 ) - ( 1 - 2 ) S 1 - 2 = Y _ 1 - Y _ 2 S 1 - 2 , wherein
, H 0 : 1 - 2 = 0 ( 1 )
[0062] In equation 1, t represents a difference between the means
(.mu..sub..gamma.1, .mu..sub..gamma.2) of the two groups in a
pooled t-test, which is used when the two groups have a similar
type of deviation. 2 Equation 2 t = ( X _ 1 - X _ 2 ) - ( 1 - 2 ) (
S 1 ) 2 n 1 + ( S 2 ) 2 n 2 ( 2 ) Equation 3 degree of freedom ( df
) = [ ( S 1 ) 2 / n 1 + ( S 2 ) 2 / n 2 ] [ ( ( S 1 ) 2 / ( n 1 - 1
) ) + ( ( S 2 ) 2 / ( n 2 - 1 ) ) ] ( 3 )
[0063] In equation 2, t represents a significant difference between
the means of the two groups in a non-pooled t-test, and in equation
3, df represents degrees of freedom. If a variance between the two
groups is high, the degrees of freedom are increased, and then the
difference between the means is analyzed. Thus, the significant
difference between the means is affected by the difference in
variances.
[0064] After performing the pooled t-test or the non-pooled t-test
depending on the difference in variances, a p-value is calculated,
based on the result of the pooled or non-pooled t-test (operation
(S825). If the p-value is at a significant level, the detected spot
is judged as an error spot (operation (S835)). For example, if the
p-value is at least 0.05, the p-value is judged as being at
significant level and the detected spot is classified as an error
spot. The value of 0.05, which is used as a criterion for the
judgment of the significance level, can be somewhat changed
depending on the results of statistical experimental results.
[0065] FIG. 9 is a block diagram illustrating a system for
detecting an error spot according to an embodiment of the present
invention.
[0066] Referring to FIG. 9, the system for detecting an error spot
is composed of a data input part (900), a variance analysis part
(910), a mean verifying part (920) and an error spot judging part
(930). The mean verifying part (920) is composed of a pooled t-test
part (922) and a non-pooled t-test part (924) operating
corresponding to the difference in variances.
[0067] The data input part (900) receives a file including the
results of the quantification operation. In addition, the data
input part (900) extracts input data which are necessary to detect
an error spot, from the file. In an embodiment of the present
invention, since analyzing variances and verifying means are
performed to detect the error spot, the respective mean, standard
deviation, and pixel number for background intensity and foreground
intensity are extracted to obtain the input data from the file.
[0068] In the variance analysis part (910), analysis of the
difference in variances for the background intensity and the
foreground intensity is performed based on a standard deviation of
the input data extracted in the data input part (900). The analysis
of the variance is performed using the f-test.
[0069] In the mean verifying part (920), verification is performed
whether the mean of the background intensity and the mean of the
foreground intensity are significantly different from each other,
based on the difference in variances in the variance analysis part
(910). The verification is performed using the t-test. The variance
analysis part (920) may perform the pooled t-test in a pooled
t-test part (922) or the non-pooled t-test in a non-pooled t-test
part (924), depending the difference in variances.
[0070] For example, if the resulting value in the f-test is at
least 0.05, the difference in variances are judged as having a
significance, and the non-pooled t-test is performed. If the
resulting value in the f-test is no more than 0.05, the pooled
t-test is performed.
[0071] In the error spot judging part (930), the p-value is
calculated based on the results in the mean verifying part (920)
and a judgment on an error spot is performed based on the p-value.
For example, if the p-value is at least 0.05, the detected spot is
classified as an error spot.
[0072] FIGS. 10 and 11 are diagrams illustrating the ratio and the
type of error spots detected in each DNA chip.
[0073] Referring to FIG. 10, 0.7 to 8.23% of the spots are detected
as error spots. As a result of analyzing the data detected as error
spots, while in most error spots, the standard deviation of the
foreground intensity (fsd) and the standard deviation of the
background intensity (bsd) are high and the foreground intensity
(fmd) and the background intensity (bmd) are low, some spots having
a high standard deviation of the intensity may be detected as error
spots even though their intensities are more than 10000.
[0074] FIG. 12 is a diagram illustrating a change of Robust M
caused by excluding error spots.
[0075] Referring to FIG. 12, with respect to the change of Robust
M, the difference is no more than about 2.5. This is a great
difference, taking it into account that if the difference is at
least 1 in the analysis, the kernel discriminating the difference
changes greatly. Thus, reliability on the results may be
increased.
[0076] The invention can also be embodied as computer readable
codes on a computer readable recording medium. The computer
readable recording medium is any data storage device that can store
data which can be thereafter read by a computer system. Examples of
the computer readable recording medium include read-only memory
(ROM), random-access memory (RAM), CD-ROMs, magnetic tapes, floppy
disks, optical data storage devices, and carrier waves (such as
data transmission through the Internet). The computer readable
recording medium can also be distributed over network coupled
computer systems so that the computer readable code is stored and
executed in a distributed fashion.
[0077] According to an embodiment of the present invention, spots
having high difference in variances for the foreground intensity
and the background intensity are detected as error spots (such as,
spots having low intensity resulting from small spot size or
incorrect alignment, or spots having partially saturated intensity)
and excluded, and thus in the subsequent statistical analysis,
errors in discriminating between a sample from a normal person and
a sample from a patient can be decreased. That is to say, the
reliability in statistical analysis can be increased.
[0078] While this invention has been particularly shown and
described with reference to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. The preferred embodiments should be considered in
descriptive sense only and not for purposes of limitation.
Therefore, the scope of the invention is defined not by the
detailed description of the invention but by the appended claims,
and all differences within the scope will be construed as being
included in the present invention.
* * * * *